Terpenes and terpenoids are found in most organisms. Their important commercial value, which is constantly increasing, is linked to the diverse range of bioactivities and functionalities encompassed by different terpenes. Accordingly, many vitamins, hormones, insect repellents, drugs, flavors and fragrances are found amongst this very large class of compounds, which all are made starting from units of 5 carbons called isoprene units.
Terpenes can be classified by the number of isoprene units present in their structure: monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), tetraterpenes (C40) and polyterpenes (Cn, n equal to or greater than 45 carbons). The plant kingdom contains a high diversity of mono- and sesquiterpenes representing thousands of different structures.
The chemical synthesis of higher terpenes such as sesqui- and diterpenes is very complex and environmentally acceptable processes for their preparation have not yet been realized. Therefore, it is a first objective of the present invention to provide methods for efficiently producing or accumulating specific terpenes while avoiding multiple-step chemical synthesis.
Studies on the biosynthetic pathway of terpenes revealed that the common C5-precursor to all terpenes is isopentenyl diphosphate (IPP). Two distinct pathways for IPP biosynthesis coexist in the plants. The mevalonate pathway (MVA) is found in the cytosol in association with the endoplasmic reticulum and the non-mevalonate pathway, also called deoxyxylulose or methyl-D-erythritol phosphate pathway (DOXP/MEP) is found in the plastids of higher plants. The starting products, the enzymes involved and the catalysed reactions are different in both pathways, and, in the cells of higher plants they operate in parallel and complement each other. Accordingly, the MVA pathway in the cytoplasm is responsible for the biosynthesis of sterols, sesquiterpenes, and polyterpenes, whereas the plastid (MEP pathway) provides C5-units for the synthesis of monoterpenes, diterpenes, for example kaurene (C20), and polyterpenes, for example carotenoids (C40) and plastoquinone-9 (C45).
Following the synthesis of IPP, it is repetitively condensed by prenyl transferases (PRT) to form the acyclic prenyl diphosphate terpene precursors for each class of terpenes, that is, geranyl-diphosphate (GPP) for the monoterpenes, farnesyl-diphosphate (FPP) for the sesquiterpenes, geranylgeranyl-diphosphate (GGPP) for the diterpenes. These precursors in turn serve as substrate for the terpene synthases or cyclases, which are specific for each class of terpene, e.g. monoterpene, sesquiterpene or diterpene synthases. Terpene synthases can catalyze complex multiple step cyclizations to form the large diversity of carbon skeleton of the terpene compounds.
Attempts have been made to isolate specific terpene synthases and WO 2004/031376 reports the isolation of the genes encoding cubebol, valencene and germacrene synthases. When E. coli cells were transformed with plasmids containing these genes, the corresponding fragrance compounds could be found in the cultivating medium. Generally, in view prior art concerned with heterologous expression of terpene synthases, it is an objective to provide means and methods for accumulating specific terpenes in still higher amounts.
In U.S. Pat. No. 5,589,619, U.S. Pat. No. 5,365,017, U.S. Pat. No. 5,349,126 and U.S. Pat. No. 5,349,126 processes for increasing squalene and sterol accumulation in transgenic plants are disclosed. These references, however, are silent as to how the accumulation of other classes of terpenes, such as mono-sesqui- and diterpenes could be increased.
The preparation of transgenic plants is also the subject of U.S. Pat. No. 6,841,717, which relates to genes associated with the MEP-pathway. This reference teaches a DNA molecule encoding an HMBPP-Synthase (GCPE protein), which was linked to a chloroplast transit peptide and was thus used to produce a transgenic plant. While this reference deals with the accumulation of tocopherol substrates, it is silent how other terpene compounds can effectively be accumulated.
In U.S. Pat. No. 6,653,530 a method for increasing carotenoid production in seed is disclosed, in which a host plant is transformed with nucleic acid sequence of Erwinia uredora encoding a phytoene synthase.
WO 00/22150 discloses methods creating or enhancing resistance to insects in plants by expressing the monoterpenes synthases limonene-, carveol and S-linool synthases in plants transformed with nucleotide sequences encoding these enzymes.
WO 02/33060 A2 discloses nucleic acid sequences and methods for producing plants and seeds having altered tocopherol content and compositions.
In WO 91/13078 DNA sequences encoding various enzymes of Erwinia herbicola are disclosed. Transformed host organisms producing GGPP and various carotenoids are also mentioned.
EP 1 063 297 provides cDNA sequences coding for farnesyl diphosphate synthase and transgenic plants expressing heterologous farnesyl diphosphate synthase.
WO 02/064764 discloses isolated or recombinent nucleic acid sequences capable of synthesizing a monoterpene linalool and/or a sesquiterpene nerolidol when contacted with the respective precursor. In example 11, the difficulty of producing sesquiterpenes in transgenic plants is acknowledged, and no concrete results in this respect are presented.
Similarly, in a publication of Aharoni et al. “Terpenoid Metabolism in Wild-Type and Transgenic Arabidobsis Plants”, the Plant cell, Vol. 15, 2866-2884, only very low levels of nerolidol were synthesized by linalool/nerolidol synthase targeted to the plastids.
The present inventors address the problem of producing or accumulating a specific, selected terpene. Preferably, a method is provided, which is suitable to produce not only a pre-determined, but any terpene of interest. The objective is thus to provide a system which allows, for example, the accumulation of any of the above-indicated sesquiterpenes, such as cubebol, valencene, germacrene, patchoulol, but which is also suitable to accumulate other terpenes. This problem has so far not been solved by the prior art, the latter basically suggesting recombinant organisms having modified properties in the MVA or MEP pathway, and observing that certain terpene end products get accumulated.
A further objective of the present invention is to provide means for generating any selected terpene, preferably a sesquiterpene, in a stereochemically pure form and with a reliable and cost effective production platform.
An important problem addressed by the present inventors is the increased accumulation of a selected terpene, preferably a sesquiterpene, in plants. In the prior art, yields of a maximum of about 10 μg terpene per g fresh weight plant material are reported. In particular with respect to sesquiterpenes, accumulation remains particularly low, in general in the order of micro-grams or below. It is thus an objective of the present invention to provide a possibility of accumulating more significant amounts of any terpene at the choice of the skilled person in a plant, and in particular of a sesquiterpene. Preferably, the plant can easily be cultivated. It is a further objective to accumulate the terpene in plant organs that provide a high biomass with respect to the total weight of the adult plant.
Another objective of the present invention is to provide plants accumulating sufficient amounts of terpenes for inhibiting growth of plant pathogens and attack by herbivores.